盐生杜氏藻锰超氧化物歧化酶(MnSOD)在大肠杆菌中的功能鉴定  

Identification of the function of superoxide dismutase gene from Dunaliella salina in SOD deleted strain K12

作　　者：邓婷婷[1] 李静[1] 马根[1] 王晓林[1] 赵向丽[1] 张飞伟[1] 乔代蓉[1] 曹毅[1] 

机构地区：[1]四川大学生命科学学院四川省生物信息与代谢工程共享实验平台,成都610064

出　　处：《四川大学学报（自然科学版）》2007年第1期176-180,共5页Journal of Sichuan University(Natural Science Edition)

基　　金：教育部新世纪人才(NCET-05-0785);国家自然科学基金(3.470928;30671125)

摘　　要：将极端耐盐的盐生杜氏藻(Dunaliella salina)的锰超氧化物歧化酶(DsMnSOD)基因克隆到表达载体pET32a中,并转入SOD缺陷型大肠杆菌菌株K12(SOD-)中,诱导重组蛋白表达,在耐盐、抗辐射和抗寒方面对其功能进行验证.结果显示,导入外源DsMnSOD基因的工程菌,在有氧条件下受到各种胁迫时,生长状况明显优于原菌,其耐受NaCl浓度从8%提高到10%,耐受紫外线(295 nm,83μW/cm2)照射时间从45 s提高到65 s,4℃培养的存活率从10.7%提高19.0%,且SOD的酶活表达依赖于Mn2+的存在.The recombinant plasmid pET32a-DsMnSOD was constructed by subcloning the ORF of DsMnSOD gene from Dunaliella salina, which is a unicellular alga possessing extremely effective mechanisms for tolerating such osmotic stress, into the pET32a vector. Then the recombinant plasmid was transformed into E. coli K12, a double mutant devoid of Mn-SOD and Fe-SOD activities. The gene was induced to express proteins and the function of the superoxide dismutase gene DsMnSOD was identified. Analysis of the results suggests that： in different stress, comparing the growing situation and living condition as aerobes in LB culture medium between K12pET32a-DsMnSOD, K12pET32a and K12 shows that the former is better. It indicated that DsMnSOD has been successfully for expressed in K12 to have enhanced its tolerance to salt （NaCl concentration from 8% to 10% ）、ultraviolet radiation（time from 45s to 65s）、cold（4℃ 10d,livability from 10.7% to 19% ） and oxygen. In addition, the activity of Mn-SOD also depends on Mn^2＋.

关 键 词：盐生杜氏藻 锰超氧化物歧化酶 K12(SOD^-) SOD酶活 

分 类 号：Q81[生物学—生物工程]